The syntrophins are a family of adapter proteins that bind numerous signaling proteins and link them to the dystrophin protein complex (DPC). All five syntrophins share a common domain structure: a PDZ domain (which binds signaling proteins), two PH domains and a C-terminal region unique to the syntrophins (the SU domain). Syntrophin-associated signaling proteins include ion and water channels, neuronal nitric oxide synthase (nNOS), protein and lipid kinases, transporters and G-protein coupled receptors. This proposal will focus on the regulation of nNOS and aquaporin-4 (AQP4) interaction with alpha-syntrophin in skeletal muscle. Recent studies have shown that nNOS is critically important to muscle function and health. Loss of nNOS increases the susceptibility of muscle to exercise-induced fatigue, perhaps due in part to defective vasoregulation. The goal of this proposal is to determine the molecular mechanisms that regulate the interaction of alpha-syntrophin with dystrophin (via the PH2-SU domains) and with nNOS and AQP4 (via the PH1-PDZ domains). Based on recent studies showing that phosphorylation/dephosphorylation of beta2-syntrophin regulates interaction with a key PDZ ligand, we propose a comprehensive approach to identify phosphorylation sites on alpha- syntrophin and determine if phosphorylation status regulates syntrophin binding to dystrophin and to its PDZ ligands. Mutants of alpha-syntrophin that mimic or prevent phosphorylation will be analyzed for interaction with dystrophin and nNOS biochemically and in cell culture. Ultimately, mutated alpha-syntrophins will be studied in vivo in transgenic mice. These studies include nNOS targeting and physiological function of skeletal muscle. New transgenic results demonstrate that the PH1 domain is important in the association of nNOS with the DPC and the sarcolemma. We will examine the role of phosphatidylinositol lipids and PH1 binding proteins in this regulatory mechanism. The DPC is capable of binding four syntrophins, thus forming a scaffold for functionally interdependent signaling proteins. Deciphering the role of phosphorylation and phosphatidylinositol binding in this process is essential to understanding the signaling function of the DPC and its role in muscle diseases.